U.S. patent number 9,778,256 [Application Number 11/922,108] was granted by the patent office on 2017-10-03 for biochip substrate and biochip.
This patent grant is currently assigned to HIPEP LABORATORIES, NIPPON LIGHT METAL COMPANY, LTD.. The grantee listed for this patent is Kiyoshi Nokihara, Yasuo Oka. Invention is credited to Kiyoshi Nokihara, Yasuo Oka.
United States Patent |
9,778,256 |
Nokihara , et al. |
October 3, 2017 |
Biochip substrate and biochip
Abstract
A biochip substrate which is free from cross-contamination due
to spot spreading or contact with spots adjacent to each other, and
a biochip using the same. A biochip substrate on which multiple
valleys for immobilizing biological substances are formed so as to
prevent cross-contamination due to spot spreading or contact with
spots adjacent to each other, and a biochip using the same are
provided. Moreover, it is found out that a desired binding in a
target molecule contained in a test sample occurs at a detectable
level in a solution system even in the case where a valley have
such a small capacity as 1 nL to 10 nL.
Inventors: |
Nokihara; Kiyoshi (Kyoto,
JP), Oka; Yasuo (Fuji, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Nokihara; Kiyoshi
Oka; Yasuo |
Kyoto
Fuji |
N/A
N/A |
JP
JP |
|
|
Assignee: |
HIPEP LABORATORIES (Kyoto-shi,
JP)
NIPPON LIGHT METAL COMPANY, LTD. (Tokyo, JP)
|
Family
ID: |
37532398 |
Appl.
No.: |
11/922,108 |
Filed: |
June 16, 2006 |
PCT
Filed: |
June 16, 2006 |
PCT No.: |
PCT/JP2006/312119 |
371(c)(1),(2),(4) Date: |
December 13, 2007 |
PCT
Pub. No.: |
WO2006/135045 |
PCT
Pub. Date: |
December 21, 2006 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20100130380 A1 |
May 27, 2010 |
|
Foreign Application Priority Data
|
|
|
|
|
Jun 17, 2005 [JP] |
|
|
2005-177466 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/54393 (20130101); B01L 3/5085 (20130101); B82Y
30/00 (20130101); B01L 2300/0893 (20130101); B01L
2300/0829 (20130101); B01L 2300/0636 (20130101); B01L
2300/12 (20130101); B01L 2300/0896 (20130101); B01L
2200/12 (20130101); C40B 60/14 (20130101) |
Current International
Class: |
C40B
60/14 (20060101); G01N 33/543 (20060101); B82Y
30/00 (20110101); B01L 3/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
|
2003-517149 |
|
May 2003 |
|
JP |
|
2003-202343 |
|
Jul 2003 |
|
JP |
|
2004-226384 |
|
Aug 2004 |
|
JP |
|
2005-017073 |
|
Jan 2005 |
|
JP |
|
2005-043312 |
|
Feb 2005 |
|
JP |
|
2005-043312 |
|
Feb 2005 |
|
JP |
|
2005-049101 |
|
Feb 2005 |
|
JP |
|
WO 98/22541 |
|
May 1998 |
|
WO |
|
WO 2004/019025 |
|
Mar 2004 |
|
WO |
|
Other References
Young et al. (Monitoring enzymatic reactions in nanoliter wells,
2003, Journal of Microscopy, vol. 212, pp. 254-263). cited by
examiner .
Tanga et al., JP 2005/043312, English translation, 18 pages, Feb.
2005. cited by examiner.
|
Primary Examiner: Boesen; Christian
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
The invention claimed is:
1. A substrate for biochips, which substrate has a plurality of
recesses formed therein for immobilizing a biologically relevant
substance(s), each of said recess having a volume of 1 nL to 10 nL,
each of said recess having an inner wall and a bottom made of
carbon, wherein functional groups for immobilizing the biologically
relevant substance(s) are bonded only to the carbon of the inner
wall and the bottom, and wherein the biologically relevant
substance(s) are immobilized to the inner wall and the bottom of
the substrate without spots that are diffused and without
cross-contamination due to contacts between adjacent spots.
2. The substrate according to claim 1, wherein said substrate is
made of a metal and a carbon layer is formed only in the inside of
each of said recesses.
3. The substrate according to claim 2, wherein said metal is
selected from the group consisting of aluminum, titanium, stainless
steel and alloys containing at least one of the metals
mentioned.
4. The substrate according to claim 3, wherein said metal is
aluminum or an alloy thereof, and a plated layer or a layer of
oxide of said metal is formed between said substrate and said
carbon layer.
5. The substrate according to any one of claims 2 to 4, wherein
said carbon layer is made of graphite, diamond, diamond-like carbon
or amorphous carbon.
6. The substrate according to claim 1, wherein said substrate is
made of carbon.
7. The substrate according to claim 6, wherein said substrate is
made of graphite or amorphous carbon.
8. The substrate according to claim 1, wherein said functional
groups are amino groups, aldehyde groups, carboxyl groups,
sulfhydryl groups or epoxy groups, or poly-lysine non-covalently
bound to said carbon layer.
9. A biochip comprising said substrate according to claim 1, and a
biologically relevant substance(s) immobilized on said
substrate.
10. A method of producing a biochip, said method comprising the
steps of providing said substrate for biochips, according to claim
1; and immobilizing a biologically relevant substance(s) on said
substrate.
Description
TECHNICAL FIELD
The present invention relates to a biochip on which a biologically
relevant substance(s) such as nucleic acids, peptides, sugars and
the like is(are) immobilized, as well as to a substrate
therefor.
BACKGROUND ART
It is well known that biochips having a flat substrate surface on
which DNAs or proteins are immobilized include those prepared by
Affymetrix method in which oligonucleotides are synthesized on the
surface of the substrate using photolithography, and those prepared
by Stanford method in which preliminarily provided probe DNAs or
probe proteins are spotted so as to immobilize them on the surface
of the substrate. Either type of the biochips is used such that
fluorescence is detected after biological reactions with a target,
and identification of the molecule or diagnosis is performed from
the resulted pattern.
Among the above-mentioned two methods, the Affymetrix method has a
drawback in that stable immobilization and synthesis of a long
oligonucleotide are difficult because the oligonucleotide is
synthesized on the surface of the substrate, and that the cost is
also high. On the other hand, in the Stanford method, in order to
place small spots of probe DNAs, probe proteins and the like are
placed on the surface of the substrate and to immobilize the
molecules to be recognized by adsorption or covalent bonds, amino
groups, aldehyde groups, silanol groups or epoxy groups are
covalently attached to, or polylysine is noncovalently attached to
the surface of the substrate. However, it is known that since these
functional groups or the polylysine are attached to the entire
surface of the substrate, spots may be diffused,
cross-contamination may occur due to contacts between adjacent
spots, and the amounts of the immobilized molecules may differ when
some spotting methods are used. It is true that uniformity in the
amount and the shape (e.g., the diameter of the spots) of the spots
is not attained due to the properties of the molecules per se, such
as the hydrophobicity and ease of ionization thereof. In recent
years, DNA chips are widespread and most of them use a glass as the
material of the substrate. However, the amount of the molecules
which can be bound by the modification of the silanol groups on the
surface of the glass is small, and when a slide glass, a generally
used substrate, is used, the amount is several nanomoles, so that
the capacity of the substrate to immobilize the molecules is low.
In case of immobilizing the molecules by adsorption, there is also
a drawback in that non-specific adsorption strongly occurs, so that
the fluorescent substances in unreacted areas, which remain even
after washing after the biological reactions, decrease the S/N
ratio of the detection.
Patent Literature 1: JP 2001-128683 A
Patent Literature 2: Japanese Translated PCT Patent Application
Laid-open No. 2005-510440
DISCLOSURE OF THE INVENTION
Problems which the Invention Tries to Solve
An object of the present invention is to provide a substrate for
biochips with which spots are not diffused and cross-contamination
due to contacts between adjacent spots does not occur.
Means for Solving the Problems
After intensive study, the present inventors inferred that a
substrate for biochips with which spots are not diffused and
cross-contamination due to contacts between adjacent spots does not
occur may be provided by forming a plurality of recesses therein
for immobilizing a biologically relevant substance(s), thereby
completing the present invention.
That is, the present invention provides a substrate for biochips,
which substrate has a plurality of recesses formed therein for
immobilizing a biologically relevant substance(s). The present
invention also provides a biochip comprising the substrate
according to the present invention, and a biologically relevant
substance immobilized on the substrate. The present invention
further provides a method of producing a biochip, the method
comprising the steps of providing the substrate for biochips,
according to the present invention; and immobilizing a biologically
relevant substance(s) on the substrate. The present invention still
further provides use of the substrate for biochips, according to
the present invention, for the production of a substrate for
biochips.
Effects of the Invention
By the present invention, a substrate for biochips and a biochip
using the substrate, with which spots are not diffused and
cross-contamination due to contacts between adjacent spots does not
occur, were provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an arrangement of the recesses formed in the substrate
prepared in an Example of the present invention.
FIG. 2 shows the structure of the peptide prepared in an Example of
the present invention.
FIG. 3 shows the relationship between the concentration of
calmodulin and fluorescence intensity measured in Example 3 of the
present invention.
FIG. 4 shows the relationship between the concentration of
calmodulin and fluorescence intensity measured in Example 4 of the
present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
As described above, the substrate for biochips according to the
present invention has a plurality of recesses for immobilizing a
biologically relevant substance(s). The number of the recesses per
one substrate is not restricted, and it is usually about 100 to
50,000, preferably about 1000 to 10,000 per one substrate having a
size of a slide glass. The volume of each recess is not restricted,
and is preferably 1 nL to 10 nL. The fact that the desired binding
of the target molecules in a test sample occurs at a detectable
level in solution systems even if the volume of each recess is so
small as mentioned above was not known, and was first discovered by
the present invention. Since the volume of each recess is small,
the amount of the test sample subjected to the reaction is also
small, which is advantageous because the volume of the valuable
test sample can be made small. The volume of each well in a
microplate, in which binding reactions are carried out in solution
systems is 200 .mu.L, to 300 .mu.L. Thus, the present inventors
first discovered that the desired binding of target molecules in a
test sample can be detected in solution systems in a volume of
1/10,000 or less of the well of a microplate. Although the size of
each recess is not restricted, when the shape of the recess is
cylindrical, one having a diameter of 50 .mu.m to 350 .mu.m, and a
depth of about 10 .mu.m to 200 .mu.m, which gives a volume of the
above-described about 1 nL to 10 nL is preferred. Although the
shape of the recess is not restricted at all, cylindrical one is
preferred from the viewpoint of ease of production.
The inner wall (including the bottom) of the recess is preferably
made of carbon such as graphite, diamond, diamond-like carbon or
amorphous carbon. To such a carbon, functional groups useful for
immobilizing the biological substances can be bound easily at a
high density by the methods described below.
In a preferred mode in which the inner wall of the recess is made
of carbon, the substrate is made of a metal, and a carbon layer
such as one made of graphite, diamond, diamond-like carbon or
amorphous carbon is formed only in the inside of the recess. In
this case, as the metal for constituting the substrate, a metal
selected from the group consisting of aluminum, titanium, stainless
steel and an alloy containing at least one of these metals is
preferred because of the reasons that these metals are easy to
work, excellent in flatness because they are rigid, excellent in
smoothness after polishing because the surface hardness is high,
and so on.
If the surface of the substrate body made of a metal is bent or
irregular, diffuse reflection is increased or the focusing in the
detection cannot be attained, so that the S/N ratio in the
detection is decreased. Therefore, the substrate body is preferably
flat and its surface is preferably smooth. Therefore, it is
preferred to anneal the substrate body under pressure to eliminate
the strain and to promote the flatness after sizing such as
punching, and, after grinding the surface to make it smooth, to
increase the surface precision by further polishing the surface.
These workings for attaining flatness and smoothness can be carried
out by conventional metal working methods. In cases where the metal
is aluminum or an aluminum alloy, since it is difficult to secure
surface precision because the metal is soft, it is preferred to
perform a hardening treatment such as electroless NiP plating or
anodic oxidation. The surface roughness Ra of the substrate body is
preferably less than 1 nm. Although the lower limit of Ra is not
restricted, about 0.2 nm is usually close to the limit of working
precision. The surface flatness of the substrate body is preferably
less than 5 .mu.m. The thickness of the substrate body is not
restricted, and is usually about 0.5 mm to 2 mm. In cases where the
substrate body is made of aluminum or an aluminum alloy, and a
plated layer of NiP or the like is formed on the substrate body, or
an oxide layer is formed on the substrate body by anodic oxidation
of the surface, the thickness of the plated layer or the oxide
layer is not restricted and is usually about 5 .mu.m to 30
.mu.m.
The recesses can be formed in the substrate made of a metal by
mechanical processing using a microdrill; laser processing using
carbon dioxide laser, YAG laser, excimer laser or the like; energy
radiation processing using focused ion beam or the like;
lithography processing; press working or the like.
In cases where a carbon layer is formed in the inside of each
recess in the substrate made of a metal, the carbon layer is a
layer made of carbon such as graphite, diamond, diamond-like carbon
or amorphous carbon, and can be formed by sputtering method, vapor
deposition method, CVD (chemical vapor deposition method) or the
like. That is, the graphite layer can be formed by, for example,
vacuum vapor deposition method using graphite particles as a vapor
deposition source. The diamond layer can be formed by, for example,
low pressure gas-phase synthesis method using a CVD apparatus
having a heat filament. The diamond-like carbon can be formed by,
for example, ion-sputtering method or high frequency plasma CVD
method. Amorphous carbon can be formed by, for example, high
frequency sputtering method. These methods can easily be carried
out using commercially available apparatuses. As described above,
in cases where the plated layer or oxide layer is formed, the
carbon layer is formed thereon. That is, the carbon layer is formed
on the surface of the substrate body indirectly through another
layer.
In cases where the carbon layer is formed in the inside of each
recess in the substrate made of a metal, it is preferred that the
carbon layer be formed only in the inside of each recess and be not
formed on the substrate surface between the recesses. As described
below, functional groups for immobilizing the biologically relevant
substance(s) can be bound to the carbon layer. If the carbon layer
is formed only in the inside of each recess and is not formed on
the substrate surface between the recesses, the functional groups
bound to the carbon layer also exist only in the recesses, so that
it is assured that the biologically relevant substance(s) be
immobilized only in the recesses. Forming the carbon layer only in
the recesses can be attained by, for example, forming the carbon
layer on the entire surface of the substrate by the above-described
sputtering method, vapor deposition method, CVD method or the like,
and then removing the carbon layer formed on the substrate surface
between the recesses by grinding the carbon layer. By this method,
the carbon layer can be easily formed only in the inside of the
small recesses.
Another preferred method for forming the inner wall of the recesses
with carbon is to make the entire substrate with carbon. By forming
the substrate itself with carbon such as graphite or amorphous
carbon, the inner wall of the recesses can be formed with carbon.
In this case, the recesses can be formed by mechanical processing
using a microdrill; laser processing using carbon dioxide laser,
YAG laser, excimer laser or the like; energy radiation processing
using focused ion beam or the like; lithography processing;
injection molding; stamping with a number of hard needles, or the
like. Even in cases where the entire substrate is made of carbon,
the substrate surface is preferably flat, and preferably has a
surface roughness within the range described above for the
substrates made of a metal. Such a surface roughness can be
attained by grinding the surface with a commercially available
grinder. The grinding is performed after attaching the functional
groups on the entire substrate to remove the functional groups
formed on the substrate surface between the recesses.
The carbon constituting the inner wall of the recesses preferably
has functional groups for immobilizing the biologically relevant
substance(s). The functional groups can be provided by binding the
functional groups to the carbon material constituting the inner
wall of the recesses. Examples of the functional groups include,
but not limited to, amino group, aldehyde group, carboxyl group,
sulfhydryl group and epoxy group. Among these groups, amino group
is especially preferred because it is versatile and binding with
biologically relevant substances is easy. These functional groups
to be covalently bound to the carbon can be covalently bound to the
carbon by cleaving C--C bond, C.dbd.C bond and/or C--O bond of the
carbon by irradiation with plasma or ultraviolet light, and binding
the resulting carbon radical with the functional groups or a
compound(s) having the functional groups. For example, amino groups
can be, as will be described in detail later in the Examples below,
covalently bound to carbon by converting the oxygen in the air to
ozone and reacting the resulting ozone with the carbon by
irradiating the carbon layer with ultraviolet light in the air,
then after evacuation, reacting chlorine gas with the resultant to
chlorinate the carbon, and, after evacuation, reacting ammonia gas
with the resultant to aminate the carbon. Alternatively, amino
groups can also be directly introduced by irradiation with ammonia
plasma. Still alternatively, amino groups can be generated on the
surface by generating radicals by irradiating the substrate surface
with argon plasma, converting the radicals to peroxide by air
oxidation, and by reacting the resulting peroxide with allylamine
or the like. Aldehyde groups can be obtained by, for example,
converting the surface of the carbon to an acid chloride, and
reducing the resulting acid chloride. Carboxyl groups may be
obtained by, for example, converting amino groups to diazonium
ions, converting the resulting diazonium ions to nitrile, and
hydrolyzing the resulting nitrile. Carboxyl groups can also be
obtained by oxidizing alkyl groups with potassium permanganate or
the like. Sulfhydryl groups can be obtained by, for example,
halogenating the surface of the carbon with light or the like, and
reacting the generated halogenated alkyl with a thiol. Epoxy groups
may be generated by treating the carbon-carbon double bonds with a
peracid. Any of these reactions may be carried out based on the
reactions in the field of organic synthetic chemistry, which are
well-known by those skilled in the art. The functional groups are
not necessarily bound to carbon by covalent bonds, but a
compound(s) having the functional group(s) can be noncovalently
attached by physical adsorption. For example, amino groups may be
given to the carbon layer by physically adsorbing poly-lysine to
the carbon layer, which poly-lysine is obtained by polycondensation
of lysine which is an amino acid having an amino group in its side
chain. The density of the functional groups given to the carbon
layer is not restricted, and usually about 10 pmol to 1000 pmol,
preferably about 100 pmol to 300 pmol per 1 cm.sup.2 of the carbon
layer.
By immobilization of a biologically relevant substance(s) to the
recesses of the above-described substrate for biochips, according
to the present invention, a biochip can be obtained. Examples of
the biologically relevant substances include nucleic acids such as
DNAs and RNAs; various proteins, antibodies, enzymes and synthetic
and natural peptides; saccharides such as polysaccharides and
oligosaccharides; various lipids; and complexes thereof
(glycoproteins, glycolipids, lipoproteins and the like). Further,
cells can also be immobilized, so that the cell is also included
within the scope of the term "biologically relevant substance".
Still further, low molecular compounds such as coenzymes, antigen
epitopes and haptens are also included within the scope of the term
"biologically relevant substance" because they specifically
interact with biopolymers such as enzymes and antibodies. These
biologically relevant substances may be bound to the
above-described carbon layer as they are, or they may be bound to
the above-described carbon layer in the state of being immobilized
to other carriers such as plastic beads.
Immobilization of the biologically relevant substance(s) to the
carbon material having the functional groups may be carried out by
well-known methods through the above-described functional groups.
For example, in cases where the functional groups are amino groups,
as will be described in detail in the Examples below, biologically
relevant substances may be immobilized to the substrate by
converting the amino groups to the corresponding anhydride with
bromoacetic acid and carbodiimide; reacting the resultant with
amino groups to bromoacetylate the surface; and reacting the
resultant with sulfhydryl groups in the biologically relevant
substances such as peptides. Alternatively, the biologically
relevant substances can be immobilized through glutaraldehyde by
reacting the amino groups with the amino groups in the biologically
relevant molecules. In cases where the functional groups are
aldehyde groups, immobilization of the biomolecules desired to be
immobilized can be attained by the reaction with the amino groups
in the biomolecules. In cases where the functional groups are
carboxyl groups, an ester is formed with N-hydroxysuccinimide, and
the resulting ester can be bound with the amino groups in the
biologically relevant substances. In cases where the functional
groups are sulfhydryl groups, immobilization may be attained by
selectively bromoacetylating the amino groups in the biologically
relevant molecules. Alternatively, immobilization may be attained
by binding the sulfhydryl groups with other sulfhydryl groups
through disulfides. Further, sulfhydryl groups can be bound by
selectively converting the amino groups at the site to be subjected
to the immobilization to maleimide, and binding the resultant with
the sulfhydryl groups (for example, N-6 maleimide caproic acid is
condensed with the amino groups). In cases where the functional
groups are epoxy groups, the biologically relevant substances may
be immobilized, similarly, by reaction of the epoxy groups with
biologically relevant substance having maleimides.
Not only in cases where, needless to say, the inner wall of the
recesses does not have the functional groups, but also in cases
where the inner wall of the recesses have the functional groups, it
is not necessary to immobilize the biologically relevant
substance(s) by covalent bonds (see Example 3 below). By simply
placing a solution(s) of the biologically relevant substance(s) to
the recesses, and drying the solution(s) to adhere the biologically
relevant substance(s) in the recesses, the substrate can be used as
a biochip. In this case, a biologically relevant substance(s) which
change(s) its(their) fluorescence or the like by binding with a
target substance(s) is(are) placed in the recesses. By using such a
dry type biochip, a small amount of a test sample can be detected
simply. Unlike the measurements in a solution, by performing the
measurements after drying the solution to be measured, and by
drying the solution of a biologically relevant substance(s) which
is a test solution, the chip can be transported and stored.
Further, when carrying out the measurement, by drying the substrate
after performing the binding reaction between the biologically
relevant substance(s) dissolved by adding a test sample solution,
and performing the measurement, a simple measurement method with
which the evaporation of the small amount of solution is not cared
can be realized. Still further, the substrate may be reused.
The present invention will now be described more concretely by way
of examples. However, the present invention is not restricted to
the Examples.
Example 1
1. Production of Substrate for Biochips (Part 1)
A high purity Al--Mg alloy plate (Mg content: 4% by weight) with a
thickness of 1.2 mm was sized to 26 mm.times.76 mm by punching with
a press. A plurality of the plates were stacked and annealed under
pressure under an atmosphere at 340.degree. C., thereby removing
strain and attaining a flatness of not more than 5 .mu.m.
Thereafter, working of the end faces and chamfer (specifically,
angle 45.degree., a length: 0.2 mm) was performed to prepare plates
with a size of 25 mm.times.75 mm. Then each plate was ground with a
double side grinding machine 16B produced by SpeedFam, in which a
sponge grindstone was mounted, to attain a thickness of 0.98 mm and
a degree of parallelization of not more than 1 .mu.m. Then micro
recesses with a size shown in FIG. 1 were formed in the substrate
by mechanical processing with a microdrill using a marking press
CAMM-3 produced by Roland. The resulting plate was then subjected
to, in the order mentioned, degreasing, etching, acid activation,
and zincate treatments. More particularly, the plate was
sequentially immersed in alkaline degreasing liquid AD-68F
(50.degree. C.) produced by Uyemura for 5 minutes, in sulfuric
acid-phosphoric acid etching liquid AD-101F (80.degree. C.) for 2
minutes, in nitric acid activating liquid (20.degree. C.) for 1
minute, and in zincate liquid AD-301F3X (20.degree. C.) for 30
seconds, thereby carrying out pretreatments. Thereafter, the plate
was immersed in electroless NiP liquid NI-422 (90.degree. C.)
produced by Meltex Corporation for 2 hours to form a plated layer
on both sides of the plate, each of which had a thickness of 12
.mu.m. Each of the plated layers was polished by 2 .mu.m with a
double side polishing machine 16B produced by SpeedFam using
colloidal silica abrasive to obtain a plate having ultrasmooth
surfaces. The plate had a thickness of 1.00 mm and a surface
roughness Ra of 0.35 nm. The flatness, degree of parallelization
and Ra were measured using a flat meter FT-SOLD produced by
Mizojiri, roundness measuring machine Talyrond produced by Rank
Taylor Hobson and stylus-type surface roughness meter Talystep
produced by Rank Taylor Hobson, respectively.
An amorphous carbon layer was then formed on one surface of the
plate using high frequency sputtering apparatus CFS-8EP produced by
Tokuda Seisakusho. Particularly, sputtering was carried out for 5
minutes under Ar atmosphere at 1.0 Pa, with a feed traveling wave
power (Pf) of 1 kW, and with a reflected wave power (Pr) of 20 W.
Thereafter, the surface was polished with a double side polishing
machine 16B produced by SpeedFam using colloidal silica abrasive to
remove the functional groups other than the inner wall of the
recesses. Then functional groups were given to the thus formed
amorphous carbon layer in the micro recesses. The functional groups
were given by the following method: First, the substrate was set in
a stainless steel vessel having a window made of a synthetic
quartz, and irradiated with an ultraviolet lamp (lamp output power:
110 W) from a distance of 3 cm, which lamp emits an ultraviolet
light having a component with a wavelength of 185 nm at 30%
intensity and a component with a wavelength of 254 nm at 100%
intensity, thereby subjecting the surface of the substrate to an
ozone treatment. After evacuation, chlorine was then introduced to
perform chlorine treatment (25.degree. C., 5 minutes) under
chlorine atmosphere at 13 Pa. Further, after evacuation, ammonia
was introduced and amination treatment (25.degree. C., 5 minutes)
was carried out under ammonia atmosphere at 13 Pa. The amount of
the amino groups on the substrate was 4.1 nmol/both surfaces. The
amount of the amino groups was measured by a method in which the
surfaces of the substrate were treated with hydrochloric acid and
then the remaining hydrochloric acid was back titrated with aqueous
sodium hydroxide solution (Japanese Patent Application No.
2005-069554).
Example 2
2. Production of Substrate for Biochips (Part 2)
After placing a thermosetting phenol resin in a mold, a two-step
heat treatment at 90.degree. C. and 120.degree. C. was performed to
prepare a Bakelite block. A plate having a thickness of 2 mm and a
size of 31 mm.times.95 mm was cut out from the block, and was
ground with a double side polishing machine 16B produced by
SpeedFam, in which an iron surface plate was mounted, to attain a
thickness of 1.30 mm and a degree of parallelization of not more
than 1 .mu.m. After chamfering, the resulting plate was slowly
heated to 1200.degree. C. thereby carbonizing the substrate to
amorphous carbon. Thereafter, using a LD-excited YVO4 laser
produced by Fuji Electric, the small recesses having the size shown
in FIG. 1 were formed in the air, and then functional groups were
attached by the following method: First, the substrate was set in a
stainless steel vessel having a window made of a synthetic quartz,
and irradiated with an ultraviolet lamp (lamp output power: 110 W)
from a distance of 3 cm, which lamp emits an ultraviolet light
having a component with a wavelength of 185 nm at 30% intensity and
a component with a wavelength of 254 nm at 100% intensity, thereby
subjecting the surface of the substrate to an ozone treatment.
After evacuation, chlorine was then introduced to perform chlorine
treatment (25.degree. C., 5 minutes) under chlorine atmosphere at
13 Pa. Further, after evacuation, ammonia was introduced and
amination treatment (25.degree. C., 5 minutes) was carried out
under ammonia atmosphere at 13 Pa. The amount of the amino groups
on the substrate was 4.1 nmol/both surfaces. The amount of the
amino groups was measured by a method in which the surfaces of the
substrate were treated with hydrochloric acid and then the
remaining hydrochloric acid was back titrated with aqueous sodium
hydroxide solution (Japanese Patent Application No. 2005-069554).
The surface of the resulting substrate was polished with a double
side polishing machine 16B produced by SpeedFam using colloidal
silica abrasive to remove the functional groups other than only the
inner wall of the recesses, thereby preparing a selectively
adsorptive amorphous carbon substrate.
Example 3
Calmodulin was measured using a peptide chip having recesses in
which a peptide having .alpha.-helix structure was immobilized,
which peptide was labeled with different fluorescence labels at its
both ends. More specifically, this was carried out as follows:
The sequence of the core region of the peptide having .alpha.-helix
structure was designed by molecular modeling using a computer
(molecular modeling using Insight II/Discover of Molecular
Simulation, U.S.) based on the amino acid sequence of the peptide
described in a reference (K. T. O'Neil and W. F. DeGrado, Trend
Biochem Sci, 15, 59-64 (1990)). As a result, the designed amino
acid sequence of the core region was
Leu-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu (SEQ ID
NO:1). This sequence is known to specifically bind to calmodulin.
To this sequence, a Cys residue as an anchor for immobilization,
and as fluorescently labeled residues, Lys(TAMRA) and Lys(FAM) were
added. Thus, an amino acid sequence
Cys-Lys(TAMRA)-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Ly-
s(FAM)-NH.sub.2 (SEQ ID NO:2) was synthesized. Fluorescent amino
acid derivatives were prepared by introducing a fluorescent group
to the side chain of lysine, and were used as building blocks for
the synthesis. That is, the fluorescently modified amino acid
derivatives were converted to N-hydroxysuccinimide active ester
derivatives using diisopropylcarbidiimide. The amino group in the
side chain of Fmoc-Lys-OH (Novabiochem, Switzerland, Product No.
04-12-1042) and the above-described active ester were reacted by a
conventional method in dimethylformamide overnight at room
temperature with stirring, and then the reaction mixture was
concentrated and subjected to precipitation with ether to obtain
Fmoc-Lys(TAMRA)-OH. Yield: 80%. (Fmoc: fluorenyl-methylcarbonyl).
In the similar manner, Fmoc-Lys(FAM)-OH was obtained. Yield:
85%
By using these fluorescent derivatives as building blocks, the
synthetic peptide shown in FIG. 2 having the labels A and B was
synthesized by the conventional solid phase peptide synthesis by
Fmoc method in a scale of 15 micromoles each. That is, Fmoc-amino
acid derivatives were sequentially polycondensed using TentaGel
SRAM, Rapp Polymere, Germany, Product No.: S30-023 (Rink amide
resin with polyethylene glycol chain) as the solid support. More
specifically, the synthesis was carried out by the method described
in Japanese Patent No. 2007165 directed to a multiple product
chemical reaction apparatus, using a commercially available
multiple peptide synthesizer, Model PSSM-8, Shimadzu
Corporation.
The synthesized labeled peptide was dissolved in 60% DMF to a
concentration of 1.0 and the obtained solution was placed in each
recess in the substrate prepared in Example 1 or Example 2 using
SpotBot (TeleChem International, U.S.) in an amount of 1.8 nL per
recess, thereby placing the fluorescently labeled .alpha.-peptide
(the substance to be recognized) in the recesses. In this case, the
peptide was not immobilized. After adding the peptide solution to
each recess and drying the solution, quantification of the binding
(recognition) was tried. Calmodulin was dissolved in a solution
containing 20 mM Tris-HCl, 100 .mu.M CaCl.sub.2, 150 mM NaCl and 20
mM PEG2000, and the resulting solution was placed in each recess in
an amount of 3.9 nL (0.5 mg/mL) per recess. The substrate was
incubated at 25.degree. C. and immediately the measurement with a
fluorescent scanner (CRBIO IIe produced by Hitachi Soft
Engineering) was carried out (excitation wavelength: 498 nm,
measurement wavelength: 579 nm).
The results of the measurement on the substrate prepared in Example
1 are shown in FIG. 3. The fluorescence changed depending on the
calmodulin concentration, so that it was proved that quantification
of calmodulin can be attained using this biochip. Similar results
were obtained also when the substrate of Example 2 was used.
Example 4
As the peptide to be immobilized to the functional groups on the
carbon layer in the recesses of the biochip prepared in Example 1,
a fluorescently labeled peptide having the following sequence was
chemically synthesized:
TABLE-US-00001 (SEQ ID NO: 3)
Ac-Cys-Gly-Lys(FAM)-Gly-Leu-Lys-Lys-Leu-Leu-Lys-
Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Lys(TAMRA)-Gly- NH.sub.2.
The fact that this peptide has .alpha.-helix structure was
confirmed by CD spectrum. Here, both "FAM" and "TAMRA" are
fluorescent dyes. When FAM is excited with light, the excitation
energy of FAM is transferred to TAMRA depending on the distance
between FAM and TAMRA, and TAMRA emits fluorescence (called
fluorescence resonance energy transfer, FRET-fluorescence). When a
protein binds to the peptide, the helix structure of the peptide is
fixed, so that FRET florescence is increased. FRET is a phenomenon
that energy is transferred from a donor molecule (FAM in this case)
in the excited state to an acceptor molecule (TAMRA in this case)
in the ground state, and fluorescence from the acceptor is
observed. The peptide is known to specifically bind to calmodulin
(CaM). Upon binding to CaM, the distance between FAM and TAMRA is
decreased. The larger the amount of CaM, the higher the measured
fluorescence intensity from TAMRA, so that the binding can be
quantified.
The above-described labeled peptide was dissolved in 60%
dimethylformamide (DMF) to a concentration of 2 .mu.M. On the other
hand, the amino groups on the substrate prepared in Example 1 were
bromoacetylated. Specifically, this was carried out as follows:
Bromoacetic acid (BrAcOH, Tokyo Chemical Industry, Mw=138.95, 2.00
mmol, 278 mg) and dicyclohexylcarbodiimide (DCC) (Aldrich
Mw=206.33, 1.00 mmol, 206 mg) were dissolved in tetrahydrofuran
(3.33 ml), and the resulting solution was gently shaken at room
temperature for 60 minutes to form bromoacetic anhydride. The
generated insoluble urea was removed by filtration. The resulting
aminated substrate was immersed in 5% solution of
diisopropylethylamine in NMP and lightly rinsed. The filtrate
obtained above was added to this substrate and the substrate was
immersed therein at room temperature for 1 hour while lightly
shaking the substrate sometimes, thereby attaining bromination. The
resulted substrate was washed with ultrapure water (Milli-Q water
(trademark)), and dried under nitrogen. The above-described labeled
peptide solution was spotted on the substrate to react the peptide
with the above-described bromoacetylated amino groups, thereby to
immobilize the peptide on the substrate. The spotting was carried
out using SpotBot apparatus produced by TeleChem International
(California, U.S.) and using a microspotting pin also produced by
TeleChem International.
To the thus prepared labeled peptide-immobilized substrate,
solutions containing different amounts of CaM (CaM was dissolved in
100 .mu.M calcium chloride solution) were applied, and fluorescence
was measured using a scanner (CRBIO IIe produced by Hitachi
Software Engineering).
The results are shown in FIG. 4. As shown in FIG. 4, the measured
fluorescence intensity increased dependently on the amount of CaM.
Thus, it was proved that a substance which specifically reacts with
the biologically relevant substance immobilized on the chip can be
quantified by the biochip according to the present invention.
Example 5
Three milligrams of (N-(6-maleimidocaproyloxy)succinimide))
produced by Dojin (hereinafter referred to as "EMCS") was weighed,
and dissolved in a 2:4:4 mixed solution of dimethylsulfoxide
(DMSO)/dimethylformamide (DMF)/dioxane to a final concentration of
0.3 mg/mL, thereby preparing an EMCS solution. The aminated
substrate was immersed in this solution at room temperature for 30
minutes, thereby allowing reaction between the amino groups and
EMCS active ester. The substrate was then washed with ethanol to
remove the unreacted EMCS and reaction side products. Then the
substrate was dried under a nitrogen atmosphere to prepare a
modified substrate in which only the inside of the microwells was
maleimidated.
A peptide having .alpha.-helix structure labeled with a fluorescent
group TAMRA alone
Ac-Cys-Gly-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Lys(TA-
MRA)-Gly-NH.sub.2 (SEQ ID NO:4) was synthesized by a conventional
method. When a protein binds to this peptide, the helix structure
is changed, and fluorescence intensity is changed accordingly. This
peptide is known to specifically binds to calmodulin (CaM) which is
a protein, and the larger the amount of CaM, the higher the
fluorescence intensity of TAMRA measured, so that CaM can be
quantified.
The peptide was dissolved in DMF/pH8.0, 10 mM Tris-HCl (1:1) to a
final concentration of 2.0 .mu.M. This peptide solution in an
amount of 3.9 nL was added to the substrate using SpotBot produced
by TeleChem International (California, U.S.) and using a
microspotting pin also produced by TeleChem International. By
leaving the substrate to stand for 30 minutes, the maleimide groups
and the thiol groups in the cystein of the peptide reacted and the
peptide was immobilized. The substrate was washed with DMF/water
(1:1).
Solutions containing different amounts of CaM (CaM was dissolved in
100 .mu.M calcium chloride solution) was added to each micro recess
in an amount of 3.9 nL per recess using SpotBot produced by
TeleChem International (California, U.S). After washing the
substrate with Milli-Q water (trade name), fluorescence intensity
was measured using a scanner (CRBIO IIe produced by Hitachi Soft
Engineering).
The results shown in FIG. 3 were obtained. Thus, the measured
fluorescence intensity changed depending on the amount of CaM added
to the substrate, so that it was proved that substances which
specifically react with the biologically relevant substance
immobilized on the chip can be quantified by the biochip of the
present invention.
SEQUENCE LISTINGS
1
4114PRTArtificial SequenceSynthetic polypeptide used for binding
assay 1Leu Lys Lys Leu Leu Lys Leu Leu Lys Lys Leu Leu Lys Leu1 5
10217PRTArtificial SequenceSynthetic peptide used for binding assay
2Cys Lys Leu Lys Lys Leu Leu Lys Leu Leu Lys Lys Leu Leu Lys Leu1 5
10 15Lys320PRTArtificial SequenceSynthetic peptide used for binding
assay 3Cys Gly Lys Gly Leu Lys Lys Leu Leu Lys Leu Leu Lys Lys Leu
Leu1 5 10 15Lys Leu Lys Gly 20418PRTArtificial SequenceSynthetic
peptide used for binding assay 4Cys Gly Leu Lys Lys Leu Leu Lys Leu
Leu Lys Lys Leu Leu Lys Leu1 5 10 15Lys Gly
* * * * *